Bismuth(III) Flavonolates: The Impact of Structural Diversity on Antibacterial Activity, Mammalian Cell Viability and Cellular Uptake

Article abstract of DOI:10.1002/chem.202000562

A series of homoleptic and heteroleptic bismuth(III) flavonolate complexes derived from six flavonols of varying substitution have been synthesised and structurally characterised. The complexes were evaluated for antibacterial activity towards several problematic Gram-positive (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE)) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. The cell viability of COS-7 (monkey kidney) cells treated with the bismuth flavonolates was also studied to determine the effect of the complexes on mammalian cells. The heteroleptic complexes [BiPh(L)₂] (in which L=flavonolate) showed good antibacterial activity towards all of the bacteria but reduced COS-7 cell viability in a concentration-dependent manner. The homoleptic complexes [Bi(L)₃] exhibited activity towards the Gram-positive bacteria and showed low toxicity towards the mammalian cell line. Bismuth uptake studies in VRE and COS-7 cells treated with the bismuth flavonolate complexes indicated that Bi accumulation is influenced by both the substitution of the flavonolate ligands and the degree of substitution at the bismuth centre.

Full text of DOI:10.1002/chem.202000562

Accepted Article
Accepted Article
Title: Bismuth(III) flavonolates: the impact of structural diversity on
antibacterial activity, mammalian cell viability, and cellular uptake
Authors: Philip C. Andrews, Kirralee J Burke, Liam J Stephens, and
Melissa V Werrett
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202000562
Link to VoR: https://doi.org/10.1002/chem.202000562
01/2020

10.1002/chem.202000562
Chemistry - A European Journal
Bismuth(III) flavonolates: the impact of structural diversity
on antibacterial activity, mammalian cell viability, and
cellular uptake
Kirralee J. Burke, Liam J. Stephens, Melissa V. Werrett, and Philip C. Andrews*
K. J. Burke, Dr. L. J. Stephens, Dr. M. V. Werrett, Prof. P. C. Andrews
School of Chemistry, Monash University
Clayton, Melbourne, VIC 3800 (Australia)
Email: phil.andrews@monash.edu
Abstract: A series of homoleptic and heteroleptic bismuth(III) flavonolate complexes derived from six flavonols of varying
substitution have been synthesised and structurally characterised. The complexes were evaluated for antibacterial activity
towards several problematic Gram-positive (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and
vancomycin-resistant Enterococcus (VRE)) and two Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Cell
viability of COS-7 (monkey kidney) cells treated with the bismuth flavonolates was also studied to determine the effect of the
complexes on mammalian cell viability. The heteroleptic complexes [BiPh(L)2] (where L = flavonolate) showed good antibacterial
activity towards all of the bacteria but reduced COS-7 cell viability in a concentration-dependant manner. The homoleptic
complexes [Bi(L)3] exhibited activity towards the Gram-positive bacteria and showed low toxicity towards the mammalian cell line.
Bismuth uptake studies in vancomycin-resistant Enterococcus (VRE) and COS-7 cells treated with the bismuth flavonolate
complexes indicated that Bi accumulation is influenced by both the substitution of the flavonolate ligands and the degree of
substitution at the bismuth centre.
Introduction
The reduced efficacy of antibiotic therapy due to the emergence of drug-resistant bacterial strains has become a global
health issue.[1] The World Health Organisation (WHO) has warned against a ‘post-antibiotic’ era unless urgent action is
taken, and recommends for the health industry to invest in the development of new antibiotics.[2] The need for novel
antibacterial agents with unique mechanisms of action to overcome the resistance of current treatments has led to
renewed interest in metallodrugs.[3] Metal complexes offer utility as potential therapeutics due to the versatility available
in the oxidation state, coordination sphere, and charge of the overall complex.[4] Coordination of bioactive molecules to
metal ions can increase their aqueous solubility, cell permeability, and stability in physiological conditions.[5] These
properties, along with the structural diversity offered by modification of the coordinating ligands, can allow metal-based
drugs to exhibit unique mechanisms of action to diverse biological targets.[6]
The use of naturally-occurring bioactive molecules or their derivatives as ligands in the preparation of new metallodrugs
can offer the advantage of incorporating molecules that already have established pharmacological properties and low
systemic toxicity.[7] This is exemplified by the recent interest in the preparation of bioactive metal complexes from
curcuminoids; the polyphenols found in turmeric (Curcuma longa), which exhibit a range of pharmacological activities.[8]
Flavonols (3-hydroxyflavones, Figure 1) are a ubiquitous subclass of the phytochemical flavonoids, which are widely
present in dietary fruits and vegetables.[9] As important dietary components, naturally-occurring flavonols have been
extensively investigated for their biological properties, and have been shown to exhibit antibacterial,[10] antioxidant,[11]
anticancer[12] and anti-inflammatory[13] activity. The therapeutic properties of natural flavonols have guided medicinal
chemists to use the 3-hydroxyflavone backbone to develop libraries of synthetic flavonols with enhanced biological
activity.[14]
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Figure 1. Structure of 3-hydroxyflavone (flavonol) with atom numbering scheme
The effective chelating α-hydroxy ketone moiety in flavonols, along with their pharmacological activity, has led to the
development of metal-flavonolate complexes for biological applications.[15] A number of Ru(II),[16] Pt(II),[17] Rh(III),[18]
Os(II),[19] and Cu(II)[20] complexes with flavonolate ligands have been synthesised and evaluated for in vitro anti-cancer
activity. Extensive work on Ru(II)-arene complexes with flavonolate ligands by the Hartinger group has shown that the
flavonolate ligand largely mediates the cytotoxicity of the overall complexes.[16c] The 3-hydroxyflavonolate ligand in the
Cu(II) complex reported by Zhou and co-workers was found to act as an ionophore for facilitating Cu(II) uptake into HepG2
cells, which is subsequently reduced to Cu(I), causing antiproliferative activity by disruption of intracellular redox
homeostasis.[20] It is notable that the examples of metal flavonolates in the literature are largely limited to complexes with
transition metal ions.[15] Motivated by the desirable biological activities of these complexes, we sought to further expand
the examples of metal flavonolates to biologically relevant main-group metals.
Bismuth compounds have been used historically for the treatment of various gastrointestinal disorders, with bismuth
subsalicylate (BSS) having been available as the over-the-counter antacid medication Pepto-Bismol since 1918.[21]
Colloidal bismuth subcitrate (CBS; De-Nol) is used clinically in combination with antibiotics such as metronidazole,
clarithromycin and amoxicillin, to treat infections caused by the gut-specific bacterium Helicobacter pylori (H. pylori).[22]
Importantly, resistance of H. pylori to bismuth treatment has not been reported despite its long-term clinical use.[23]
We have previously shown that bismuth(III) compounds derived from thiolates,[24] arenesulfonates,[25] and
benzohydroxamates,[26] exhibit good in vitro antibacterial activity towards H. pylori as well as drug-resistant bacteria such
as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). Heterocyclic
bismuth(III) complexes reported by Kotani and co-workers have also shown potent antibacterial activity towards clinical
isolates of MRSA.[27] Despite the promising biological activities of novel bismuth complexes, only BSS and CBS are
currently used clinically.[21] Further expansions of such bismuth compounds may therefore lead to the development of
new clinically-relevant bismuth antibacterial drugs.
Accordingly, we have used a series of six flavonols with varying structural substitution on the phenyl (B) ring (Figure 2) to
synthesise novel bismuth(III) flavonolate complexes. Homoleptic [Bi(L)3] (where L = flavonolate) and heteroleptic
[BiPh(L)2] derivatives were synthesised to obtain additional structural diversity. The complexes were evaluated for
antibacterial activity against three Gram-positive (MRSA, VRE, Staphylococcus aureus) and two Gram-negative
(Escherichia coli, Pseudomonas aeruginosa) bacteria. The biological evaluation of the complexes was extended to
mammalian cell viability assays and bismuth uptake studies to assess structure-activity relationships and the potential
utility of bismuth flavonolate complexes as antibacterial agents.
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Figure 2. Flavonols 1-6 used in this study to prepare Bi(III) flavonolate complexes: 3-hydroxyflavone, 3-HFH (1); 4-dimethylamino-3-
hydroxyflavone, 4-DMAFH (2); 4-diethylamino-3-hydroxyflavone, 4-DEAFH (3); 4-methoxy-3-hydroxyflavone, 4-MFH (4); 3,4-dimethoxy-3-
hydroxyflavone, 3,4-DMFH (5); 3,4,5-trimethoxy-3-hydroxyflavone, 3,4,5-TMFH, (6).
Results and discussion
Six flavonols containing varying substitution on the phenyl (B) ring were first synthesised before complexation with
bismuth. Flavonols 3-hydroxyflavone (3-HFH, 1), 4-dimethylamino-3-hydroxyflavone (4-DMAFH, 2), 4-diethylamino-3-
hydroxyflavone (4-DEAFH, 3), 4-methoxy-3-hydroxyflavone (4-MFH, 4), 3,4-dimethoxy-3-hydroxyflavone (3,4-DMFH,
5), and 3,4,5-trimethoxy-3-hydroxyflavone (3,4,5-TMFH, 6) were synthesised in two steps using known literature
procedures.[28] In the first step, a Claisen-Schmidt condensation with 2-hydroxyacetophene and the respective substituted
benzaldehydes formed the corresponding substituted 2’-hydroxychalcones in yields of 60-70 %, which were used without
further purification. The 2-hydroxychalcones were then cyclised to the respective flavonols 1–6 via Algar-Flynn-Oyamada
conditions and purified by recrystallisation from ethanol (see Supporting Information). The formation of 1–6 was confirmed
by 1H and 13C NMR spectroscopy, mass spectrometry, and IR spectroscopy.
The non-nucleophilic base bismuth tert-butoxide [Bi(OtBu)3] is useful in preparing homoleptic tris-substituted Bi(III)
complexes from compounds containing an accessible acidic proton (pKa < 15).[24a, 25, 29] Consequently, [Bi(OtBu)3] was
utilised in the formation of tris-flavonolate Bi(III) complexes; [Bi(3-HF)3] 1b, [Bi(4-DMAF)3] 2b, [Bi(4-DEAF)3] 3b, [Bi(4-
MF)3] 4b, [Bi(3,4-DMF)3] 5b, and [Bi(3,4,5-TMF)3] 6b. The aforementioned complexes were synthesised following the
addition of a THF solution of the appropriate flavonol (1–6) to a THF solution of [Bi(OtBu)3] in a 3:1 stoichiometry (Scheme
1). The resulting complexes precipitated from the reaction mixture and were isolated by filtration in yields of 70–82 % after
washing with small amounts of THF and Et2O and drying in vacuo.
Scheme 1. Synthetic methods used to prepare homoleptic Bi(III) tris-flavonolate complexes 1b–6b, and heteroleptic Bi(III) monophenyl bis-
flavonolate complexes 1c–6c from flavonols 1–6.
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To increase the chemical and structural diversity through modification of the bismuth(III) coordination sphere, a series of
heteroleptic mono-phenyl Bi(III) bis-flavonolate [BiPh(L)2] complexes were also synthesised. The heteroleptic bismuth
flavonolate complexes were synthesised using a salt metathesis route whereby [BiPhCl2] was added to two equivalents
of the sodium salt of the target flavonol in dry methanol at 0 C (Scheme 1). Heteroleptic complexes [BiPh(3-HF)2] 1c,
[BiPh(4-DMAF)2] 2c, [BiPh(4-DEAF)2] 3c, [BiPh(4-MF)2] 4c, [BiPh(3,4-DMF)2] 5c, and [BiPh(3,4,5-TMF)2] 6c, were
isolated as solids from the reaction mixture and washed with small amounts of methanol and diethyl ether, and dried in
vacuo to give the target complexes in yields of 70–88 %. Complexes 1b–6b and 1c–6c are all air and moisture stable
bright yellow (1b, 1c, 4b, 4c, 5b, 5c, 6b, 6c), orange (2c, 3c), or red (2b, 3b) solids. All complexes except 1b are soluble
in most organic solvents, such as THF, toluene, DMSO, and DCM. All complexes are insoluble in water and are sparingly
soluble in ethanol and methanol. Elemental analysis of all complexes was consistent with the expected compositions.
The 1H and 13C NMR spectra of homoleptic bismuth flavonolate complexes 1b–6b and heteroleptic complexes 1c–6c
were measured in DMSO-d6 and referenced to the residual solvent peak. The hydroxyl signals present in the 1H NMR
spectra (9.10–9.46 ppm) of the free flavonols 1–6 are absent in the spectra for corresponding bismuth complexes,
confirming flavonol deprotonation and complexation to bismuth. The 1H NMR spectra of the heteroleptic complexes 1c–
6c show the expected flavonolate ligand proton signals as well as the o-, m- and p- proton signals associated with the
single phenyl ligand in the range of 8.43–8.46, 7.49–7.53, and 7.02–7.05 ppm, respectively (See Supporting information
for NMR spectra of all complexes).
The FT-IR spectra of the bismuth flavonolate complexes show a decrease in intensity of the characteristic broad –OH
band found in the spectra of the free flavonols, suggesting deprotonation and formation of the target complexes. The
spectra also show the carbonyl C=O stretching vibration of the respective flavonolate ligands in 1b–6b and 1c–6c to be
in the range 1584–1547 cm-1, which is shifted from 1611–1598 cm-1 in the spectra of the parent flavonols 1–6. The red
shift of v(C=O) in the IR spectra of flavonols upon complex formation is characteristic of metal-flavonolate complexes.[30]
A comparison of v(C=O) IR absorptions in 1b–6b, 1c–6c, and the parent flavonols 1–6, is given in Table S1 of the ESI.†
High-resolution electrospray ionisation mass spectrometry (high res ESI-MS) of 1b–6b and 1c–6c further confirm the
formation of the target complexes.
X-ray Crystallography
The solid-state structures of 1b (Figure 3), 2b (Figure S1), 5b (Figure S3), 6b (Figure S4), 1c (Figure S5), 2c (Figure S6),
5c (Figure S7), and 6c (Figure 5) were determined by single crystal X-ray diffraction on crystals grown from DMSO
solutions. In the asymmetric unit of 1b (Figure 3), three 3-hydroxyflavonate ligands bind to the Bi(III) centre through the
deprotonated hydroxyl group, with Bi–O bond lengths of 2.205(2) Å (Bi(1)–O(1) and Bi(1)–O(5)), and 2.167(2) Å (Bi(1)–
O(3)). Given the difference in ion size of Bi(III), these bond lengths correspond well with analogous M–O bonds in the 3-
hydroxyflavonolate complexes with Mn(II) (2.1274(16) Å) and Fe(III) (1.955(7) Å),[31] and are shorter than the equivalent
bond in the Pb(II) 3-hydroxyflavonolate complex (2.2270(19) Å).[32] The carbonyl oxygens of the flavonolate ligands in 1b
also coordinate to the Bi(III) centre, having longer Bi–O(=C) bond lengths in the range of 2.476(3) –2.704(2) Å. Both of
the anionic and coordinative Bi–O bond lengths in 1b are consistent with the range of reported Bi–O bond distances.[33]
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Figure 3. Asymmetric unit of the molecular structure of [Bi(3-HF)3] (1b). Thermal ellipsoids are shown at the 50% probability level. Hydrogen atoms
have been omitted for clarity. Selected bond lengths (Å) and angles (): Bi(1)–O(1), 2.205(2); Bi(1)–O(2), 2.476(3); Bi(1)–O(3), 2.167(2); Bi(1)–O(4),
2.517(2); Bi(1)–O(5), 2.205(2); Bi(1)–O(6), 2.704(2); O(1)–Bi(1)–O(2), 70.63(9); O(3)–Bi(1)–O(4), 69.33(8); O(5)–Bi(1)–O(6), 66.49(8).
The formation of five-membered O,O-chelate rings by the flavonolate ligands coordinating to the Bi(III) centre is consistent
with the structures of other metal-flavonolate complexes.[30a, 31-32] The three flavonolate O–Bi–O bite angles in 1b are in
the range of 66.49(8)–69.33(8) which is similar to the Pb(II) 3-hydroxyflavonolate complex (68.14(7)) previously
described.[32] The carbonyl C=O distances of the 3-hydroxyflavonolate ligands in 1b are in the range of 1.254(4)–1.262(4)
Å which are elongated compared to free 3-hydroxyflavone (1.232(3) Å).[34] However, the hydroxylate C–O bond lengths
(1.327(4)–1.336(4) Å) are shorter compared to the free flavonol (1.357(3) Å). The elongation and shortening of these
respective bonds is consistent with the structures of Fe(III), Mn(II), and Ni(II) 3-hydroxyflavonolate complexes.[30a, 31]
Considering the geometry around the Bi(III) centre, a seventh interaction is indicated in the direction of the empty
coordination site of the Bi(III) atom to the O(6) carbonyl oxygen of an adjacent molecule (Figure 4). The length of the
Bi(1)O(6) interaction (2.986(2) Å) is within the sum of the van der Waals radii of bismuth and oxygen.[35] Thus 1b
crystallises as a dimer with the Bi(III) centre adopting a seven-coordinate, distorted pentagonal bipyramidal geometry with
a planar 4-membered Bi2O2 ring.
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Figure 4. Dimeric structure of 1b, [Bi(3-HF)3]2. The length of the Bi(1)O(6) interaction is 2.986(2) Å. Thermal ellipsoids are shown at the 50 %
probability level. Hydrogen atoms have been omitted for clarity. Symmetry operator: (2-X, 1-Y, 2-Z).
Complex [Bi(4-DMAF)3] (2b) also crystallises as a dimer through the equivalent Bi(1)O(6) interaction, with a Bi(1)O(6)
bond length of 3.1657(15) Å. Contrasting to 1b and 2b, the Bi(III) centres in the crystal structures of [Bi(3,4-DMF)3] (5b)
and [Bi(3,4,5-TMF)3] (6b) do not make any intermolecular interactions to adjacent molecules. Due to their monomeric
structures, the Bi(III) centres in 5b and 6b contain an unoccupied void where the stereochemically active lone pair likely
sits trans to the flavonolate ligands. Complexes 5b and 6b thus adopt a six-coordinate, distorted octahedral geometry.
In the structure of the heteroleptic complex [BiPh(3,4,5-TMF)2(DMSO)] (6c(DMSO)) (Figure 5), the Bi(III) centre is six-
coordinate, with two chelating bidentate flavonolate ligands, one phenyl ligand, and one coordinating DMSO solvent
molecule. The phenyl ligand lies orthogonal to the equatorial plane containing the flavonolate ligands and coordinating
DMSO molecule, giving the complex a pentagonal pyramidal geometry around the Bi(III) centre, with an average C–Bi–
O bond angle of 85.7. The pentagonal pyramidal geometry of 6c(DMSO) is consistent with the structures of other
heteroleptic mono-phenyl Bi(III) complexes that contain a coordinating solvent molecule.[25, 36] Trans to the phenyl ligand
in 6c(DMSO) is an unoccupied void in the Bi(III) coordination sphere, likely where the stereochemically active lone-pair
lies, as is typically observed in such Bi(III) complexes.[25, 36]
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Figure 5. Molecular structure of [BiPh(3,4,5-TMF)2(DMSO)] (6c(DMSO)). Thermal ellipsoids are shown at the 50 % probability level. Hydrogen
atoms have been omitted for clarity. Selected bond lengths (Å) and angles (): Bi(1)–O(1), 2.293(3); Bi(1)–O(2), 2.556(3); Bi(1)–O(3), 2.252(3);
Bi(1)–O(4), 2.536(3); Bi(1)–O(5), 2.618(3); Bi(1)–C(1), 2.240(4). O(1)–Bi(1)–O(2), 68.04(9); O(3)–Bi(1)–O(4), 68.58(9); C(1)–Bi(1)–O(1), 84.42(11);
C(1)–Bi(1)–O(2), 88.66(11); C(1)–Bi(1)–O(3), 90.08(12); C(1)–Bi(1)–O(4), 84.42(11); C(1)–Bi(1)–O(5), 82.84(11).
The two 3,4,5-trimethoxy-3-hydroxyflavonolate ligands in 6c(DMSO) adopt a cis-configuration around the Bi(III) centre
and deviate from the equatorial plane due to the sterics of the bulky 3,4,5-trimethoxy-phenyl moieties. The flavonolate
ligands have bite angles of 68.04(9) and 68.58(9) for O(1)–Bi(1)–O(2) and O(3)–Bi(1)–O(4), respectively. These angles
are similar to those in the corresponding tris-substituted complex 6b, where the flavonolate bite angles are in the range
of 68.14(9)-70.95(9) (See Table S6). The Bi(1)–C(5) bond length of 2.240(4) Å in 6c(DMSO) is consistent with Bi–C
bond lengths reported for similar heteroleptic Bi(III) complexes of general formula [BiPh(L)2].[24a, 25, 37] The hydroxylate Bi–
O bond lengths of 2.293(3) Å (Bi(1)–O(1)) and 2.252(3) Å (Bi(1)–O(3)) are longer than the analogous bonds in the tris-
substituted flavonolate complex 6b (2.182(3)–2.207(3) Å), and longer than the hydroxylate Pb–O bond in the 3-
hydroxyflavonolate Pb(II) complex.[32] The coordinating DMSO molecule makes a significantly longer interaction (2.618(3)
Å) to the Bi(III) centre through the O(5) oxygen atom compared to the two coordinating Bi–O=C interactions made by the
carbonyl oxygens of the flavonolate ligands (2.556(3) Å and 2.536(3) Å). Complexes 1c, 2c, and 5c also crystallised with
single coordinating solvent molecule (DMSO or THF) and show isostructural geometries to 6c(DMSO).
Solid-state and solution stability
To determine if the solid bismuth flavonolate complexes were stable to air and atmospheric moisture, the 1H NMR
spectrum of each of the complexes 1b–6b and 1c–6c was measured at regular intervals over a 6-month period. The
complexes were stored as neat solids, in air and at ambient temperature during the study. The spectra obtained for each
complex did not change over this time period indicating that the bismuth(III) flavonolate complexes are stable in ambient
conditions in the solid state. As the complexes were going to be evaluated in biological assays, their respective solution-
state stability was studied, using [Bi(3,4,5-TMF)3] 6b and [BiPh(3,4,5-TMF)2] 6c as representative examples for the
homoleptic and heteroleptic series, respectively. As the bismuth flavonolate complexes have poor water solubility, DMSO
was used as the vehicle solvent to prepare stock solutions of the compounds for the assays. The stability of complexes
6b and 6c in DMSO-d6 was thus assessed over a 24 h period at room temperature and 40 ºC. After 24 h, the resulting 1H
NMR spectra of 6b and 6c in both temperature conditions remained unchanged to the initial spectra (t = 0), indicating that
the complexes are stable towards decomposition or hydrolysis in DMSO during the timeframe used for biological assays.
UV-Vis spectroscopy was then used to establish the stability of 6b and 6c in Dulbecco’s Modified Eagle Media (DMEM)
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cell culture medium containing 10 % fetal bovine serum (FBS). After 72 h, the respective absorption spectra of 6b and 6c
in the complete cell culture media showed no significant changes from the initial spectra (see Figure 6a and S8). These
results indicate that the bismuth flavonolate complexes have good stability in cell culture media in the presence of serum
proteins during the course of incubation for biological assays. To determine if 6b and 6c were stabilised by serum proteins
such as bovine serum albumin, the stability studies were repeated under the same conditions in DMEM without added
FBS. Both complexes 6b and 6c were found to precipitate from the serum-free media over time, as indicated by the
decrease in the absorption bands (See Figure 6b and S8). The discrepancy in stability between FBS-supplemented and
plain cell culture media suggests that the bismuth flavonolate complexes are maintained in solution by the serum proteins.
Figure 6. a) Absorption spectrum of 6b (40 M) in DMEM + 10% FBS over 72 h; b) absorption spectrum of 6b (40 M) in plain DMEM (no FBS)
over 12 h. The appearance of the small band at 560 nm in both spectra is due to the phenol red indicator in the DMEM becoming the di-anion from
incubating the DMEM solutions in the absence of CO2.[38]
Stability in the presence of glutathione
Glutathione (GSH) is an abundant tripeptide present in mammalian cells in relatively high intracellular concentrations of
0.5–10 mM, where it serves as a major antioxidant inhibiting cellular damage by reactive oxygen species.[39] GSH also
plays an important role in heavy metal detoxification, due in large to the affinity of soft metals for its cysteinyl thiol group,
allowing them to be transported and excreted.[40] Colloidal bismuth citrate (CBS) has been shown to react with the cysteinyl
thiol moiety of GSH both in vitro and in red blood cells to form the [Bi(GS)3] complex.[41] The formation of [Bi(GS)3] is
thought to be the reason for the low toxicity associated with bismuth drugs, whereby [Bi(GS)3] is ultimately deposited as
inert bismuth sulfide (Bi2S3) particles.[42] It was therefore of interest to investigate the stability of the bismuth flavonolate
complexes in the presence of GSH, to determine how they may behave in an intracellular environment.
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The stability of [Bi(3,4,5-TMF)3] 6b and [BiPh(3,4,5-TMF)2] 6c in the presence of GSH was first probed by UV-vis
spectroscopy. To replicate the high concentration of GSH found in the cytosol, the complexes (30 M) were exposed to
3 mM of GSH (100 mol equivalents) at pH 7 in ultrapure water. Due to the insolubility of 6b and 6c in pure water, the
complexes were dissolved in DMSO and then added to the GSH solution (final DMSO concentration: 0.8%). Addition of
6b and 6c to 3 mM GSH induced a rapid decrease in the absorption bands of the respective spectra (Figures S10 and
S11). After 24 h, the initially (t = 0) transparent-yellow solutions were colourless and contained a fine pale precipitate,
which was identified by 1H NMR as the free flavonol (6). These results suggest that 6b and 6c undergo a GSH mediated
dissociation, whereby the flavonolate ligands are protonated and released. This reactivity is consistent with that observed
for Cu(II) 3-hydroxyflavonolate [Cu(3-HF)2], which was shown to dissociate and release 3-hydroxyflavone in the presence
of GSH.[20]
The reaction of 6b and 6c with excess GSH was further studied by 1H NMR spectroscopy using 10 mol equivalents of
GSH. Due to the contrasting solubilities of the complexes and GSH, a solvent mixture of D2O + 15 % DMSO-d6 was used.
Addition of DMSO-d6 solutions of 6b or 6c to 10 equivalents of GSH in D2O resulted in rapid precipitation of the free
flavonol 6, as identified by 1H NMR. The precipitated flavonol was removed by filtration and the filtrates were analysed
immediately by 1H NMR spectroscopy. In the reaction of 6b with GSH, the 1H NMR spectrum of the filtrate shows new
glutathione signals at δ = 4.28, 4.35 and 4.70 ppm (Figure 7), which are consistent with the cysteinyl protons signals of
the [Bi(GS)3] complex as reported by Sadler.[41] The integrals of the signals in the spectrum also reflect that 3 of the 10
equivalents of GSH have reacted to form [Bi(GS)3] (Figure S12).
Figure 7. a) 1H NMR spectrum of 1.5 mM 6b in the presence of 10 mol equivalents (15 mM) GSH at pH 7 in D2O + 15% DMSO-d6 after precipitated
flavonol 6 was removed by filtration, showing appearance of new signals at δ = 4.28, 4.35 and 4.70 ppm, characteristic of the [Bi(GS)3] complex; b)
1H NMR spectrum of GSH at pH 7 in D2O + 15% DMSO-d6
There was marked differences in the 1H NMR spectrum of 6c with 10 equivalents of GSH (following the removal of the
precipitated flavonol) compared to the analogous reaction of 6b with GSH, notably that the spectrum did not show the
characteristic signals of the [Bi(GS)3] complex. There is a downfield shift in the cysteinyl CH2 signal from δ = 2.97 ppm
(multiplet) in free GSH, to a broad multiplet centred on 3.15 ppm (Figure 8), suggesting that the Bi(III) centre is interacting
with the cysteinyl sulfur atom of GSH,[43] however it is not exclusively forming [Bi(GS)3]. Significantly, the 1H NMR spectrum
suggests that the phenyl ligand in 6c remains intact and bound to the Bi centre, with the proton signals resonating at δ =
8.46 (o, d), 7.73 (m, t), and 7.43 (p, t). The integrations of the phenyl signals relative to the GSH signals is consistent with
the 1:10 ratio of 6c to GSH that was initially reacted (Figure S13). Mass spectrometry on the NMR solution showed signals
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at m/z 592.09846 and 897.17057, which correlate well with [BiPh(GS)] (calcd: 592.0955) and [BiPh(GS)2] (calcd:
897.1636). These 1H NMR studies therefore suggest that the homoleptic bismuth flavonolate complex dissociates in the
presence of excess GSH to form [Bi(GS)3], while the heteroleptic complex forms [BiPh(GS)2], due to the apparent stability
of the Bi–CPh bond. To additionally probe the stability of the Bi–CPh bond in the presence of GSH, BiPh3 was added to 10
mol equivalents of GSH in D2O/DMSO-d6. The resulting 1H NMR spectrum showed no changes in the frequency of the
protons in the phenyl groups or in GSH compared to their individual spectra recorded in the same solvent mixture (Figure
S14). Taken together, these studies suggest that the Bi–CPh bond is stable to GSH mediated dissociation, which may
have implications for bioactivity of the heteroleptic bismuth flavonolate complexes.
Figure 8. a) 1.5 mM 6c in the presence of 10 mol equivalents (15 mM) GSH at pH 7 after precipitated flavonol 6 was removed by filtration (D2O +
15% DMSO-d6). Note the presence of phenyl signals at δ = 7.43, 7.73 and 8.46 ppm; b) 1H NMR spectrum of GSH at pH 7 in D2O + 15% DMSO-
d6 highlighting the cysteinyl CH2 protons
While there are a multitude of other intracellular inorganic molecules, proteins, sugars and lipids that the bismuth
flavonolate complexes may interact with in physiological systems, these initial investigations suggest that they are likely
to undergo a GSH mediated dissociation when an excess of GSH is present, such as in the cytosol of a mammalian cell.
This reactivity would also likely extend to other intracellular thiols such metallothionein and cysteine rich peptides.[44]
Antibacterial Activity
Flavonols 1–6, homoleptic complexes 2b–6b, and heteroleptic complexes 1c–6c were evaluated for in vitro antibacterial
activity against three Gram-positive (MRSA, VRE, Staphylococcus aureus (S. aureus)) and two Gram-negative
(Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa)) bacteria using the broth microdilution method.
Complex 1b was excluded from all biological assays due to its poor solubility in DMSO. As the bismuth precursors [Bi(O-
tBu)3] and BiPhCl2 could not be included in the assays due to their moisture sensitivity, BiPh3 was included as a
phenylbismuth reference. The antibacterial activity of the compounds is expressed in terms of their minimum inhibitory
concentration (MIC), defined as the lowest concentration at which bacterial growth is visibly inhibited. The results are
listed in Table 1.
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Table 1. MIC values for flavonols 1–6, BiPh3, and bismuth flavonolate complexes 2b–6b and 1c–6c against Gram-positive bacteria MRSA, VRE,
S. aureus, and Gram-negative E. coli and P. aeruginosa (g/mL) [M].
Compound
MRSA
S. aureus
> 50
VRE
E. coli
> 50
P. aeruginosa
> 50
Flavonols 1-6
BiPh3
> 50
> 50
12.5 [28.4]
25 [23.8]
50 [44.0]
12.5 [12.4]
6.25 [5.7]
6.25 [5.2]
3.125 [4.1]
12.5 [14.7]
12.5 [13.8]
6.25 [7.6]
6.25 [7.1]
3.125 [3.3]
12.5 [28.4]
25 [23.8]
50 [44.0]
25 [24.7]
6.25 [5.7]
6.25 [5.2]
3.125 [4.1]
12.5 [14.7]
12.5 [13.8]
3.125 [3.8]
1.56 [1.7]
1.56 [1.6]
6.25 [14.2]
25 [23.8]
50 [44.0]
25 [24.7]
12.5 [11.3]
12.5 [10.5]
3.125 [4.1]
12.5 [14.7]
12.5 [13.8]
6.25 [7.6]
6.25 [7.1]
3.125 [3.3]
> 50
> 50
[Bi(4-DMAF)3]
[Bi(4-DEAF)3]
[Bi(4-MF)3]
2b
3b
4b
5b
6b
1c
2c
3c
4c
5c
6c
> 50
25 (23.8]
50 [4.0]
> 50
> 50
25 [24.7]
25 [22.7]
25 [21.0]
12.5 [16.4]
25 [29.]
[Bi(3,4-DMF)3]
[Bi(3,4,5-TMF)3]
[BiPh(3HF)2]
> 50
> 50
3.125 [4.1]
12.5 [14.7]
6.25 [6.9]
6.25 [7.6]
6.25 [7.1]
6.25 [6.6]
[BiPh(4-DMAF)2]
[BiPh(4-DEAF)2]
[BiPh(4-MF)2]
[BiPh(3,4-DMF)2]
[BiPh(3,4,5-TMF)2]
12.5 [13.8]
12.5 [15.2]
6.25 [7.1]
6.25 [6.6]
All of the bismuth flavonolate complexes (2b–6b and 1c–6c) inhibited the growth of the Gram-positive bacteria S. aureus,
VRE, and MRSA, while the flavonols 1–6 showed no activity at the highest concentration measured (50 g/mL). Given
the clinical impact of multidrug resistant bacteria, the ability of the bismuth flavonolate complexes to inhibit growth of
MRSA and VRE at low M concentrations is promising for their potential application as antibacterial agents. BiPh3 also
showed an inhibitory effect on the growth of the three Gram-positive bacterial strains with MIC values in the range 14.2–
28.4 M. The lack of activity of the parent flavonols 1–6 suggests that complexation of the flavonols with bismuth is
necessary for the observed antibacterial activity. A general trend observed was that the heteroleptic bismuth flavonolate
complexes displayed greater antibacterial activity (had lower MIC values) than the corresponding homoleptic analogues.
This result supports observations from related studies which demonstrate the seemingly important role that heterolepticity
plays in determining antibacterial activity of bismuth(III) complexes.[24a],[45],[46],[47] This effect was most pronounced in the
dialkylamino-flavonolate complexes, where the heteroleptic 2c and 3c were approximately 2- and 4-fold more active than
homoleptic 2b and 3b, respectively. The substitution of the flavonolate ligand was also shown to influence the overall
activity of the bismuth complexes. Complexes with the dialkylamino-flavonolate ligands (2b, 2c, 3b, 3c) were notably less
active than the complexes containing methoxy-substituted flavonolate ligands. In fact, 2b and 3b were less active than
the reference compound BiPh3 towards the Gram-positive bacteria. The most active complexes in both the homoleptic
and heteroleptic series were derived from the 3,4-dimethoxy-3-hydroxyflavonolate (5b, 5c) and 3,4,5-trimethoxy-3-
hydroxyflavonolate (6b, 6c) ligands, highlighting the positive role that functionalising the flavonolate ligands with additional
methoxy groups has on enhancing antibacterial activity. However complex 1c, containing the unsubstituted 3-
hydroxflavonolate ligand, was also highly active with a MIC of 4.1 M against all three Gram-positive bacteria. The MIC
value range of the bismuth flavonolate complexes towards MRSA, VRE, and S. aureus correspond well with previously
synthesised bismuth thiolate complexes.[24b]
The heteroleptic compounds 1c–6c also showed good activity towards the Gram-negative E. coli, giving MIC values in
the range of 4.1–14.7 M, inferring broad-spectrum activity for these complexes. The respective MIC values were similar
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to those for the Gram-positive species, with 1c being the most active complex towards E. coli with a MIC of 4.1 M.
Conversely, the homoleptic bismuth flavonolate complexes 2b–6b and BiPh3 did not inhibit the growth of E. coli at the
highest concentration measured (50 g/mL). The general trend of Bi(III) complexes being less active towards E. coli
compared to Gram-positive bacteria is consistent with our previous studies on bismuth thiolates[24] and phosphinates.[48]
For example, the mono-phenyl dithiolato bismuth(III) complex [BiPh(Br-PTOT)2] (Br-PTOT = 5-[2-(4-bromophenyl)thiazol-
4-yl]-1,3,4-oxadiazole-2(3H)-thione) had MIC values in the range of 1.0–1.6 M for MRSA, VRE, and S. aureus, but
showed no antibacterial activity towards E. coli at 103 M.[45] Studies by Kotani and co-workers on cyclic
organobismuth(III) compounds showed similar effects, however the MIC values improved when the E. coli was pre-treated
with polymyxin B nonapeptide; an antibacterial peptide that increases the permeability of the outer membrane of Gram-
negative bacteria.[27] Their study inferred that the poor antibacterial activity was due to the inability of the bismuth(III)
compounds to permeate the additional outer membrane of E. coli.[27] We therefore envisage that the poor efficacy of the
homoleptic bismuth flavonolate complexes 2b–6b towards E. coli is also likely due to this effect. This limitation appears
to be overcome by the substitution of one flavonolate ligand in 2b–6b with a phenyl group, as shown by the increased
antibacterial activity of the heteroleptic analogues. The lack of activity (MIC > 50 g/mL) shown by BiPh3 however,
indicates that the presence of both flavonolate and phenyl ligands are required for the observed bactericidal activity of
1c–6c against E. coli. The bismuth flavonolate complexes also showed moderate activity towards the problematic Gram-
negative bacterium P. aeruginosa, with MIC values in the range of 6.6–44.0 M, while BiPh3 displayed no activity at 50
g/mL; highlighting the requirement for the flavonolate ligands to target this particular strain. Interestingly, the homoleptic
complexes 2b–6b displayed activity towards P. aeruginosa but not towards E. coli, indicating a species-specific activity.
Overall, the bismuth flavonolate complexes derived from flavonols 1, 5, and 6 showed the greatest activity across the five
bacterial strains, with the heteroleptic complexes (1c–6c) generally being more potent than the homoleptic (2b–6b) series.
Bi uptake in VRE
It was of interest to determine the relative cellular bismuth uptake of the homoleptic complexes 2b–6b and the
corresponding heteroleptic analogues 2c–6c in bacterial cells, to understand whether the improved efficacy of the
heteroleptic complexes to inhibit bacterial growth could be due to their higher cellular accumulation. To investigate this,
the bismuth content was measured in VRE bacterial cells that were treated with 5 g/mL, (the approximate MIC of the
most active complexes), of bismuth flavonolates 2b–6b, 2c–6c and BiPh3 for 24 h. Cell lysates were subsequently
prepared by HNO3 digestion and the average Bi content was determined by inductively coupled plasma mass
spectrometry (ICP-MS). The Bi uptake was normalised to the number of VRE cells and the results are presented as ng
Bi/106 cells (Figure 9). The untreated VRE control was processed alongside the bismuth treated cells in the experiments
and had no detectable reading of bismuth.
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Figure 9. a) Bismuth uptake in VRE bacteria exposed to 5 g/mL of bismuth-flavonolates 2b–6b, 2c–6c or BiPh3 for 24 h; b) Compounds BiPh3
and 2b–6b with scaled y-axis.
VRE treated with homoleptic bismuth flavonolate complexes 2b–6b had significantly lower Bi accumulation than VRE
treated with the corresponding heteroleptic analogues 2c–6c. VRE cells incubated with BiPh3 also showed low Bi uptake
in a similar range to 2b–6b. The amount of Bi accumulation in VRE treated with bismuth flavonolate complexes is thus
largely influenced by the substitution at the bismuth centre, with the combination of two flavonolate ligands and one phenyl
ligand facilitating an increased Bi uptake. The generally lower activity of the homoleptic complexes 2b–6b towards Gram-
positive bacteria may therefore be due to reduced Bi uptake and accumulation. It is notable however that a higher amount
of Bi uptake does not necessarily correlate to a lower MIC value. VRE treated with 4c had the highest relative Bi uptake
out of all the complexes, but 4c was not the most active compound towards VRE (MICVRE = 7.6 M), with 6c being the
most potent complex (MICVRE = 3.3 M). In fact, VRE treated with 6c showed the lowest accumulation of Bi out of all the
heteroleptic complexes. The mechanism of action of the bismuth flavonolate complexes in VRE may therefore not simply
be a consequence of cellular accumulation of bismuth and its subsequent interaction with intracellular targets. Notably,
the bismuth drugs CBS and BSS have been shown to affect bacterial morphology and outer membrane integrity and of
Gram-negative H. pylori and Yersinia enterocolitica without significant internalisation of bismuth.[49] Further research will
be required to eludicate the targets of the homoleptic and heteroleptic bismuth flavonolate complexes and determine their
mechanism of antibacterial action.
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Mammalian cell viability
As the bismuth flavonolate complexes showed promising antibacterial activity, their effect on mammalian cell viability was
evaluated using COS-7 (monkey kidney) cells. The COS-7 cells were treated with varying concentrations of bismuth
flavonolates 2b–6b and 1c–6c for 24 h at 37 C. Parent flavonols 1–6 and BiPh3 were also evaluated under the same
conditions for comparison. The cell viability as a function of concentration was determined by the colorimetric CellTitre
Blue assay, whereby viable, metabolically-active cells convert resazurin to the fluorescent end product resorufin.[50] The
IC50 values defined as the concentration required to decrease cell viability to 50% of the control (untreated cells), were
calculated from dose-response curves (Figures S15–S17 in ESI) and are summarised in Table 2.
Table 2. IC50 values of BiPh3, flavonols 1–6 and bismuth flavonolate complexes 2b–6b and 1c–6c towards COS-7 cells.
Compound
IC50 (M)a
Flavonols 1-6
BiPh3
> 50
> 50
[BiL3]
2b-6b
1c
> 50
[BiPh(3-HF)2]
19.73  0.45
20.18  0.67
18.16  0.50
19.64  0.89
10.24  0.17
7.51  0.14
[BiPh(4-DMAF)2]
[BiPh(4-DEAF)2]
[BiPh(4-MF)2]
2c
3c
4c
[BiPh(3,4-DMF)2]
[BiPh(3,4,5-TMF)2]
5c
6c
a IC50 is the concentration at which the compounds inhibit 50% cell viability measured by CellTitre Blue assay after 24 hours incubation. The results
are presented as the mean ± SD of the mean from 3 independent experiments.
Flavonols 1–6, BiPh3, and homoleptic complexes 2b–4b did not reduce cell viability of COS-7 cells at the highest
concentration measured (50 M). Homoleptic complexes 5b and 6b produced some reduction in cell viability cells at
higher doses in the range of 37.5 - 50 M, however they did not reduce cell viability to 50%. Contrastingly, the heteroleptic
complexes 1c–6c reduced the cell viability of COS-7 cells in a dose-dependent manner, giving IC50 values in the range of
7.51–20.18 M. Thus concerning the preliminary bioactivity profiles of the bismuth flavonolate complexes, a general
structure-activity relationship is observed: the homoleptic complexes 2b–6b have no or moderate antibacterial activity
and low mammalian cell toxicity, while the heteroleptic complexes 1c–6c show improved antibacterial activity but also
display higher toxicity towards mammalian cells. The overall activity of the bismuth flavonolate complexes is therefore
strongly influenced by the degree of substitution at the bismuth centre. A similar effect has been observed in bismuth(III)
indole-carboxylate compounds, where the mono-phenyl bis-indole-carboxylato complexes significantly reduced cell
viability of primary human fibroblasts (IC50 values in the range of 1.38–1.43 M), while the analogous homoleptic tris-
indole-carboxylato compounds did not reduce cell viability at 100 M.[47]
The substitution of the flavonolate ligands also influenced the activity of the complexes towards COS-7 cells. In the
heteroleptic series, complexes 1c–4c all showed similar IC50 values in the range of 18.16–20.18 M, indicating that
substitution in the 4-position of the flavonolate ligand has little effect on the activity of the overall bismuth flavonolate
complex. This contrasts to studies of Ru(II)-arene flavonolate complexes, where the substituent in the 4-position of the
flavonolate ligands had a marked influence on the toxicity of the compounds towards various cancer cell lines.[16c]
Complexes 5c and 6c had the lowest IC50 values of 10.2  0.2 M and 7.5  0.1 M, respectively. These IC50 values are
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approximately 2-fold lower than complexes 1c–4c, highlighting that additional methoxy groups in the 3- and 5-position(s)
of the flavonolate ligand has influences the observed activity. This structure-activity relationship was also seen in the
homoleptic complexes, where 5b and 6b reduced COS-7 cell viability at higher concentrations while 2b–4b showed no
activity at the highest concentration tested.
The COS-7 IC50 values and MIC data from the antibacterial studies were used to calculate the selectivity indices
(SI=IC50/MIC) for the most active homoleptic (5b, 6b) and heteroleptic (5c, 6c), with the results given in Table 3.
Table 3. Selectivity indices of the bismuth flavonolates showing the greatest antibacterial activity
Selectivity indexa
Compound
MRSA
> 8.8
> 9.6
1.4
S. aureus
> 8.8
> 9.6
6.0
VRE
> 4.4
> 4.8
1.4
E. coli
P. aeruginosa
[Bi(3,4-DMF)3]
[Bi(3,4,5-TMF)3]
[BiPh(3,4-DMF)2]
[BiPh(3,4,5-TMF)2]
5b
6b
5c
6c
-
> 2.2
> 2.4
1.4
-
1.4
1.1
2.3
4.7
2.3
1.1
aSelectivity index (SI) = IC50/MIC. The IC50 value used for calculating the selectivity indices of 5b and 6b was 50 M.
Complex 5b and 6b had the highest selectivity indices of 8.8 and 9.6, respectively, for MRSA and S. aureus, inferring
good selectivity for these bacteria over mammalian cells. The lowest selectivity indices were 5c and 6c for E. coli and P.
aeruginosa (SIs = 1.1 and 1.4), resulting from their low IC50 values for COS-7 cells and reduce efficacy in targeting the
Gram-negative bacteria.
The homoleptic complexes 5b and 6b showed improved selectivity to the heteroleptic analogues 5c and 6c. This is most
pronounced for the drug-resistant MRSA strain, where the selectivity indices improve 4- and 6-fold in the homoleptic
complexes. In the case of 6b (MICMRSA = 5.2 M) and 6c (MICMRSA = 3.3 M), there is a small decrease in antibacterial
activity from exchanging a flavonolate ligand with a phenyl group but a notable improvement in selectivity. These results
suggest the homoleptic complexes may be more therapeutically useful due to their preferable toxicity profile. Future work
on bismuth flavonolates may look at structural modifications to the flavonolate ligand that improve the antibacterial activity
while maintaining a high selectivity index.
Bi uptake in COS-7 cells
To understand the observed disparity between the activity of the homoleptic and heteroleptic complexes towards
mammalian cells, Bi uptake studies in COS-7 cells were performed. COS-7 cells were incubated with 5 M of 2b–6b, 1c–
6c, and BiPh3 for 24 h. The average Bi uptake was determined by ICP-MS and the results were normalised to the number
of cells and presented as ng Bi/106 cells (Figure 10). The untreated cells control and DMSO-vehicle control which were
processed alongside the bismuth-treated cells in the experiments had no detectable reading of bismuth.
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Figure 10. Bismuth uptake by COS-7 cells exposed to 5 M of BiPh3 and bismuth flavonolate complexes 2b–6b, 1c–6c for 24 h. (*, p < 0.05
between the homoleptic complex and the corresponding heteroleptic analogue).
COS-7 cells incubated with the bismuth flavonolate complexes (2b–6b and 1c–6c) contained a statistically higher (p >
0.05) amount of Bi compared to cells incubated with BiPh3 at the same concentration, indicating that the flavonolate
ligands enhance cellular bismuth uptake compared to phenyl ligands alone. The high IC50 value of BiPh3 (IC50 > 50 M)
may therefore be due to its low uptake and hence its poor accumulation in cells. Unexpectedly, the heteroleptic complexes
2c–6c did not have a higher uptake of Bi than the respective homoleptic analogues 2b–6b. Instead, 2b–6b had either a
statistically higher (p < 0.05) Bi uptake than the corresponding heteroleptic derivative (in the case of 2c, 3c, 5c), or the
uptake was not statistically different compared to the analogous heteroleptic complex (4c, 6c). These results suggest that
higher levels of Bi accumulation in COS-7 cells do not necessarily correlate to higher toxicity (lower IC50 values). There
was also no strong correlation between the Bi uptake of the heteroleptic complexes and the respective IC50 values, except
in the case of 6c, which had the highest Bi uptake of the heteroleptic complexes and lowest IC50 value (7.5  0.1 M). It
is notable that complexes 6b and 6c had superior Bi uptake in COS-7 cells compared to 2b–5b and 2c–5c, indicating
complexation of Bi(III) with 3,4,5-trimethoxyflavonolate ligands (6) facilitates in the increased cellular Bi uptake
compared to the other substituted flavonolate ligands. Notably flavonol 6 (3,4,5-trimethoxy-3-hydroxyflavone) has well-
established metabolic stability and chemopreventive properties.[51] The relatively high uptake of complexes 6b and 6c in
COS-7 cells may therefore be due to their complexation with the highly bioactive 3,4,5-trimethoxyflavonolate ligands.
While the cellular localisation of Bi was not determined in these experiments, previous studies have shown Bi to localise
within the cytosol[52] or lysosomes[53] of mammalian cells. As the heteroleptic bismuth flavonolate complexes 1c–6c had
similar or lower Bi uptake compared to the homoleptic complexes 2b–6b , but induced markedly different outcomes on
COS-7 cell viability, it suggests that the complexes may have differing modes of action that are not necessarily a function
of the amount of cellular Bi uptake and accumulation. It is not currently known whether the complexes remain intact upon
entering the cell, however as suggested by the glutathione stability studies, the homoleptic bismuth flavonolate complexes
are likely to dissociate and form [Bi(GS)3] upon transport into the glutathione-rich cytosol. This may be a detoxification
pathway for the homoleptic complexes,[42] resulting in their observed low toxicity while having similar Bi uptake levels to
the heteroleptic complexes. Conversely, the heteroleptic complexes may undergo alternate intracellular pathways due to
the low degree of lability of the Bi-CPh bond, which ultimately leads to the associated toxicity as shown by the lower IC50
values of 1c–6c towards COS-7 cells.
Conclusion
Six flavonols 1–6 were used to prepare a series of homoleptic (1b–6b) and heteroleptic (1c–6c) bismuth(III) flavonolate
complexes with aims to investigate their potential utility as antibacterial agents. The novel complexes were fully
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characterised and the solid-state structures of complexes 1b, 2b, 5b, 6b, 1c, 2c, 5c, and 6c were determined by single
crystal X-ray diffraction. These crystal structures represent, to the best of our knowledge, the first structural examples of
bismuth flavonolate complexes. Stability studies of 6b and 6c in the presence of excess glutathione (GSH) showed that
the homoleptic complex 6b dissociates to form [Bi(GS)3] while the heteroleptic complex 6c retains its phenyl ligand and
forms [BiPh(GS)2].
The bismuth flavonolate complexes were evaluated for antibacterial activity against three Gram-positive (MRSA, S.
aureus, VRE) and two Gram-negative (E. coli, P. aeruginosa) bacterial species. The heteroleptic complexes 1c–6c
inhibited the growth of all the bacteria, with MIC values in the range 1.6–29.5 M. Homoleptic complexes 2b–6b also
showed good activity towards the Gram-positive bacteria, but were less active than the analogous heteroleptic complexes
containing the same flavonolate ligands. Complexes 2b–6b also had moderate or no antibacterial activity towards the
Gram-negative bacteria, with MIC values in the range of 21.0–44.0 M towards P. aeruginosa while exhibiting no activity
towards E. coli. Cellular Bi uptake studies in VRE bacteria treated with the bismuth flavonolate complexes showed that
the homoleptic complexes had a significantly lower Bi uptake compared to the heteroleptic analogues, highlighting that
the combination of the flavonolate ligands and phenyl ligand enhances bismuth uptake into VRE. The homoleptic
complexes 2b–6b were found to have little effect on the cell viability of COS-7 cells, having IC50 values > 50 M.
Contrastingly, the heteroleptic complexes 1c–6c reduced COS-7 cell viability in a concentration-dependent manner, giving
IC50 values in the range of 7.51–20.18 M, with 6c being the most toxic compound. Cellular Bi uptake studies in COS-7
cells showed that cells treated with the homoleptic complexes had similar or statistically higher Bi uptake compared to
cells treated with the analogous heteroleptic complexes, indicating that a high accumulation of Bi in COS-7 cells does not
necessarily correlate with increased mammalian cell toxicity. This preliminary biological study thus demonstrates that the
bioactivity of bismuth flavonolate complexes is largely influenced by the substitution of the flavonolate ligands and degree
of substitution of the bismuth centre. The heteroleptic complexes displayed good antibacterial activity but reduced the cell
viability of mammalian cells at low M values, while the homoleptic complexes were less toxic to mammalian cells but
also exhibited lower antibacterial activity. The structure-activity relationships highlighted in these findings may therefore
aid in designing more selective and effective bismuth drug compounds of this type.
Experimental section
General: All benzaldehydes, 2-hydroxyacetophenone and L-glutathione were purchased from Oakwood Chemicals and
were used without further purification. The bismuth(III) precursors [Bi(OtBu)3] and [BiPhCl2] were synthesised using
sublimed [BiCl3] and were stored in an argon filled glovebox. [Bi(OtBu)3] was synthesised through the salt metathesis
reaction of [BiCl3] and potassium tert-butoxide [K(OtBu)] (1:3 ratio) in dried THF at 0 C, with the crude solid being
extracted with dried pentane using a soxhelette apparatus which was then removed in vacuo.[54] [BiPhCl2]was synthesised
by stirring [BiPh3] and [BiCl3] in a 1:2 ratio in dried diethyl ether at room temperature for 4 hours, producing a pale-yellow
solid which was subsequently isolated and washed with small amounts of diethyl ether and dried in vacuo. All reactions
using [Bi(OtBu)3] and [BiPhCl2] were treated as moisture sensitive and were performed in oven-dried Schlenk flasks under
an atmosphere of dry nitrogen using a vacuum/nitrogen manifold and Schlenk techniques. Tetrahydrofuran (THF) was
dried by a MBraun-SPS-800 solvent purification system and stored over 4Å molecular sieves under a dry nitrogen
atmosphere before use. Methanol was distilled over CaH2 before use and stored over 3Å molecular sieves under nitrogen.
1H and 13C NMR spectra in DMSO-d6 were recorded on a Bruker DRX 600 MHz Cryo spectrometer at room temperature
using the solvent residual signals (δ = 2.50 ppm, 1H; δ = 39.52 ppm, 13C) as internal standards. Mass spectrometry (ESI)
of flavonols 1–6 were performed on a Micromass Platform QMS spectrometer with a cone voltage of 35 eV. High resolution
ESI-MS analysis of compounds 1b–6b and 1c–6c was carried out at the Monash Analytical Platform, Australia (School
of Chemistry, Monash University). Test compounds were infused directly into the MS via a kdScientific infusion pump at
a static flow rate of 650 µL/h. MS setup was as follows: Agilent 6220 TOF MS system (Santa Clara, CA, USA) with a
multimode dual nebuliser ESI/APCI source. The MS was operated in positive or negative mode using the following
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conditions: nebulizer pressure 35 psi, gas flow-rate 8 L/min, gas temperature 300°C, capillary voltage 2500/-2500 V,
fragmentor 150 and skimmer 65 V. Instrument was operated in the extended dynamic range mode with data collected in
m/z range 100-3200. Infrared spectra were measured on an Agilent Technologies Cary 630 FTIR spectrometer in the
range of 4000-500 cm-1. Infra band frequencies are reported in wavenumbers (cm-1) and intensities reported qualitatively
as strong/sharp = s, medium = m, weak = w, or broad = b. Melting points (m.p.) are uncalibrated and were measured on
a Stuart Scientific melting point apparatus SMP10 in open end capillary tubes. Elemental analysis (C, H and N) was
performed by the Campbell Microanalytical lab, Department of Chemistry, University of Otago, New Zealand, or at the
Science Centre, London Metropolitan University. The average Bi uptake in cells incubated with different bismuth
complexes was compared by paired Student’s t test (*: p<0.05).
Synthesis and characterisation
General procedure for the synthesis of tris-flavonolate Bi(III) complexes 1b–6b (GP1): The relevant flavonol (1–6)
(1.5 mmol, 3 equiv) was added to a Schlenk flask and dried under vacuum for 4 hours prior to use, before being dissolved
in 10 mL of THF. The flavonol solution was then added dropwise to [Bi(OtBu)3] (0.5 mmol, 1 equiv) dissolved in 10 mL of
THF, which resulted in an immediate colour change and precipitation. The suspension was stirred overnight. The
precipitate was collected via gravity filtration and washed with small amounts of THF and diethyl ether, and then dried in
vacuo to yield the tris-substituted Bi(III) flavonolate complex.
[Bi(3-HF)3] (1b): The title compound was prepared according to GP1 using 1 (1.5 mmol, 357 mg). Yield: 344 mg (0.37
mmol, 74 %), bright yellow powder; m.p. > 300 C; 1H NMR (600 MHz, DMSO-d6): δ 8.38 (d, J = 7.7 Hz, 2H; H2), 8.12-
8.03 (m, 1H; H5) 7.81-7.77 (m, 2H; H7, H8 ), 7.43 (ddd, J = 8.2, 6.0, 2.0 Hz, 1H; H6), 7.18 (t, J = 7.4 Hz, 1H; H4), 6.99 (t, J
= 7.7 Hz, 2H; H3); FT-IR ṽ = 3054 (w), 2113 (w), 1931 (w), 1610 (w), 1547 (s), 1508 (s), 1461 (m), 1441 (m), 1409 (s),
1351 (m), 1309 (m), 1229 (m), 1207 (s), 1150 (s), 1115 (m), 1033 (m), 904 (m), 751 (s), 684 (s), 659 (s); HRMS (ESI)
([Bi(L)3+Na]+) calcd. for C45H27BiO9Na m/z: 943.1357 found: 943.1231; Elemental analysis calcd (%) for BiC45H27O9: C
58.71, H 2.96; found: C 58.51, H 2.74. Due to the very poor solubility of 1b, a 13C NMR spectrum could not be obtained.
[Bi(4-DMAF)3] (2b): The title compound was prepared according to GP1 using 2 (1.5 mmol, 422 mg). Yield: 430 mg (0.41
mmol, 82 %), bright red powder; m.p. 280 C (decomp); 1H NMR (600 MHz, DMSO-d6): δ 8.25 (d, J = 9.1 Hz, 2H; H2),
8.07 (dd, J = 8.2, 1.4 Hz, 1H; H5), 7.75, (dd, J = 8.4, 0.8 Hz, 1H; H8), 7.70 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H; H7), 7.41 (ddd, J
= 8.2, 6.9, 0.8 Hz, 2H; H6), 6.20 (d, J = 9.1 Hz, 2H; H3), 2.79 (s, 6H; -CH3); 13C NMR (151 MHz, DMSO-d6): δ 176.8 (C4),
153.5 (C9), 153.2 (C4), 150.3 (C2), 147.5 (C3), 132.4 (C7), 128.9 (C2), 124.2 (C6, C5), 122.5 (C10), 119.1 (C8), 117.9 (C1),
110.8 (C3), 39.7 (-CH3); FT-IR: ṽ = 2891 (w), 2798 (w), 2113 (w), 1605 (m), 1583 (m), 1561 (m), 1543 (m), 1494 (s), 1475
(s), 1398 (s), 1357 (s), 1250 (m), 1191 (s), 1110 (s), 992 (m), 905 (m), 814 (s), 754 (s), 668 (s); HRMS (ESI) ([Bi(L)3+Na]+)
calcd. for C51H42BiO9N3Na m/z: 1072.2623 found: 1072.2585; Elemental analysis calcd (%) for BiC51H42O9N3: C 58.35, H
4.03, N 4.00; found: C 58.26, H 4.03, N 3.87.
[Bi(4-DEAF)3] (3b): The title compound was prepared according to GP1 using 3 (1.5 mmol, 464 mg). Yield: 462 mg (0.40
mmol, 80 %), orange-red powder; m.p. 283 C (decomp); 1H NMR (600 MHz, DMSO-d6): δ 8.13 (d, J = 9.2 Hz, 2H; H2),
8.10 (dd, J = 8.0, 1.5 Hz, 1H; H5) 7.74 (dd, J = 8.5, 1.4 Hz, 1H; H8), 7.71 (ddd, J = 8.5, 6.7, 1.5 Hz, 1H; H7), 7.42, (ddd, J
= 8.0, 6.7, 1.4, 1H; H6), 6.07 (d, J = 9.2 Hz, 2H; H3), 3.14 (q, J = 7.0 Hz, 4H; -CH2), 0.94 (t, J = 7.0 Hz, 6H; -CH3); 13C
NMR (151 MHz, DMSO-d6): δ 176.5 (C4), 153.7 (C9), 153.5 (C4), 147.8 (C2), 146.3 (C3), 132.3 (C7), 129.2 (C2), 124.3
(C5, C6), 122.7 (C10), 118.0 (C8), 117.8 (C1), 110.0 (C3), 43.6 (-CH2), 12.4 (-CH3); FT-IR: ṽ = 2962 (w), 2119 (w), 1602
(w), 1584 (m), 1566 (m), 1491 (s), 1477(s), 1394 (s), 1345 (s), 1270 (m), 1252 (m), 1192 (s), 1145 (m), 1111(m), 1011(m),
907 (m), 816 (s), 756 (s), 725 (s), 668 (s); HRMS (ESI) ([Bi(L)3+Na]+) calcd. for C57H54BiO9N3Na m/z: 1156.3562 found:
1156.3544; Elemental analysis calcd (%) for BiC57H54O9N3: C 60.37, H 4.80, N 3.71; found: C 60.30, H 4.69, N 3.81.
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[Bi(4-MF)3] (4b): The title compound was prepared according to GP1 using 4 (1.5 mmol, 402 mg). Yield: 359 mg (0.35
mmol, 70 %), bright yellow powder; m.p. 299-301 C (decomp); 1H NMR (600 MHz, DMSO-d6): δ 8.33 (d, J = 8.9 Hz, 2H;
H2), 8.06 (dd, J = 7.9, 1.3 Hz, 1H; H5), 7.77, (dd, J = 8.3, 1.0 Hz, 1H; H8), 7.74 (ddd, J = 8.3, 6.9, 1.4 Hz, 1H; H7), 7.42
(ddd, J = 7.9, 6.9, 1.0 Hz, 1H; H6), 6.49 (d, J = 8.9 Hz, 2H; H3), 3.62 (s, 3H; O-CH3); 13C NMR (151 MHz, DMSO-d6): δ
178.1 (C4), 159.8 (C9), 153.8 (C4), 151.5 (C2), 147.3 (C3), 133.1 (C7), 129.1 (C2), 124.5 (C5), 124.4 (C6), 122.3 (C10), 118.2
(C8), 113.3 (C3), 55.0 (-OCH3); FTIR: ṽ = 2960 (w), 2836 (w), 2109 (w), 1919 (w), 1605 (m), 1591 (m), 1547 (s), 1498 (s),
1401 (s), 1352 (m), 1298 (s), 1248 (s), 1202 (s), 1148 (s), 1109 (s), 1022 (m), 832 (s), 750 (s), 671 (m); HRMS (ESI)
([Bi(L)3+Na]+) calcd. for C48H33BiO12Na m/z: 1033.1674 found: 1033.1454; Elemental analysis calcd (%) for BiC48H33O12:
C 57.04, H 3.29; found: C 56.89, H 3.30.
[Bi(3,4-DMF)3] (5b): The title compound was prepared according to GP1 using 5 (1.5 mmol, 477 mg). Yield: 405 mg (0.36
mmol, 72%), bright yellow powder; m.p. 295 C (decomp); 1H NMR (600 MHz, DMSO-d6): δ 8.04 (m, 3H; H2, H6, H5),
7.79 (d, J = 8.4 Hz, 1H; H8), 7.76 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H; H7) 7.43 (ddd, J = 7.9, 6.9, 0.9 Hz, 1H; H6), 6.48 (d, J =
8.7 Hz, 1H; H5), 3.66 (s, 3H; 3-OCH3), 3.41 (s, 3H; 4-OCH3); 13C NMR (151 MHz, DMSO-d6): δ 178.1 (C4), 153.7 (C9),
151.3 (C4), 149.7 (C3), 147.9 (C2), 147.7 (C3), 132.9 (C7), 124.5 (C5), 124.4 (C6), 124.3 (C1), 122.2 (C6), 121.3 (C10),
118.2 (C8), 110.7 (C 5), 110.6 (C2), 55.2 (3-OCH3), 54.9 (4-OCH3); FTIR: ṽ = 3014 (w), 2835 (w), 2288 (w), 2107 (w),
1589 (m), 1577 (m), 1554 (s), 1499 (s), 1480 (s), 1462 (s), 1399 (s), 1311 (m), 1265 (m), 1204 (s), 1174 (s), 1141 (s),
1024 (s), 1007 (s), 880 (w), 855 (m), 815 (m), 761 (s), 708 (m), 670 (m); HRMS (ESI) ([Bi(L)3+Na]+) calcd. for
C51H39BiO15Na m/z: 1123.1990 found: 1123.1971; Elemental analysis calcd (%) for BiC51H39O15: C 55.64, H 3.57; found:
C 55.55, H 3.51.
[Bi(3,4,5-TMF)3] (6b): The title compound was prepared according to GP1 using 6 (1.5 mmol, 492 mg). Yield: 452 mg
(0.38 mmol, 76%), bright yellow powder; m.p. 180 C (decomp); 1H NMR (600 MHz, DMSO-d6): δ 7.96 (m, 1H; H5), 7.85-
7.74 (m, 4H; H2, H6, H7, H8), 7.43 (ddd, J = 8.3, 6.2, 1.8 Hz, 1H; H6), 3.61 (s, 3H; 4-OCH3), 3.42 (s, 6H; 3-OCH3, 5-
OCH3); 13C NMR (151 MHz, DMSO-d6) δ 178.5 (C4), 153.7 (C9), 152.2 (C3, C5), 150.1 (C2), 148.5 (C3), 138.4 (C4), 133.1
(C7), 127.2 (C1), 124.3 (C5), 124.2 (C6), 122.1 (C10), 118.3 (C8), 105.0 (C2, C6), 60.1 (4-OCH3), 55.3 (3-OCH3, 5-OCH3);
FTIR: ṽ = 2936 (w), 2832 (w), 1735 (w), 1578 (m), 1550 (m), 1483 (m), 1459 (m), 1396 (s), 1335 (m), 1291 (m), 1240 (m),
1197 (m), 1173 (m), 1125 (s), 1055 (m), 999 (m), 887 (m), 862 (m), 754 (s); Elemental analysis calcd (%) for BiC54H45O18:
C 54.46, H 3.81; found: C 54.39, H, 3.70.
General procedure for the synthesis of bis-flavonolate mono-phenyl Bi(III) complexes 1c–6c (GP2): The relevant
flavonol 1–6 (1 mmol, 2 equiv) and NaOH pellets (1 mmol, 2 equiv) was added to a Schlenk flask and dried under vacuum
for 4 hours prior to use. Dried methanol (15 mL) was then added to the flask resulting in the formation of a brightly-
coloured suspension which was then left to stir for 1 hour. The flask was then placed in an ice bath for 10 minutes before
adding [BiPhCl2] (0.5 mmol, 1 equiv) to the suspension via a solid addition tube. The resulting suspension was allowed to
warm to room temperature and stirred for a further 2 hours. The precipitate was then collected via gravity filtration and
washed with small amounts of methanol and diethyl ether, and then dried in vacuo to yield the bis-flavonolate mono-
phenyl Bi(III) complex.
[BiPh(3-HF)2] (1c): The title compound was prepared according to GP2 using 1 (1.0 mmol, 238 mg). Yield: 268 mg (0.35
mmol, 70 %), bright yellow powder; m.p. 265 C (decomp); 1H NMR (600 MHz, DMSO-d6): δ 8.81 (dd, J = 7.9, 1.5 Hz 4H;
H2), 8.46 (dd, J = 7.9, 1.1 Hz, 2H; Ho), 8.10, (dd, J = 8.1, 1.3 Hz, 2H; H5), 7.81 (d, J = 8.4 Hz, 2H; H8), 7.81 (ddd, J = 8.4,
6.9, 1.6 Hz, 2H; H7), 7.55-7.50 (m, 10 H; Hm, H4, H3) 7.46 (ddd, J = 7.9, 6.9, 1.0 Hz, 2 H, H6) 7.05 (tt, J = 7.3, 1.2 Hz, 1H;
Hp); 13C NMR (151 MHz, DMSO-d6): δ 179.3 (C4), 153.9 (C9), 149.7 (C2), 149.6 (C3), 135.0 (Co), 133.4 (C7), 133.0 (C1),
131.4 (Cm), 129.0 (C4), 128.5 (C2), 127.1 (Cp), 124.5 (C3), 124.4 (C5), 124.3 (C6), 121.8 (C10), 118.3 (C8); FTIR: ṽ = 3066
(w), 1614 (w), 1549 (s), 1509 (m), 1481 (m), 1406 (m), 1350 (m), 1309 (m), 1208 (s), 1149 (m), 1112 (m), 1076 (m), 993
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(m), 905 (m), 750 (s), 723 (s), 689 (s), 675 (m); HRMS (ESI) ([BiPh(L)2+Na]+) calcd. for C36H23BiO6Na m/z: 783.1196
found: 783.1187; Elemental analysis calcd (%) for BiC36H23O6: C 56.85, H 3.05, found: C 56.92, H 3.15.
[BiPh(4-DMAF)2] (2c): The title compound was prepared according to GP2 using 2 (1.0 mmol, 281 mg). Yield: 313 mg
(0.37 mmol, 74 %), bright orange powder; m.p. 182 C; 1H NMR (600 MHz, DMSO-d6): δ 8.72 (d, J = 9.1 Hz, 4H; H2),
8.42 (dd, J = 7.9, 1.2 Hz, 2H; Ho), (d, 8.04 (dd, J = 8.0, 1.4 Hz, 2H; H5), 7.75 (d, J = 8.4 Hz, 2H; H8), 7.68 (ddd, J = 8.4,
6.9, 1.4 2H; H7), 7.49 (t, J = 7.6 Hz, 2H; Hm), 7.40 (ddd, J = 7.9, 6.9, 0.8 Hz, 2H; H6), 7.02 (tt, J = 7.3, 1.0 Hz, 1H; Hp),
6.78 (d, J = 9.1 Hz, 4H; H3), 2.98 (s, 12H, N-CH3); 13C NMR (151 MHz, DMSO-d6): δ 177.2 (C4), 153.3 (C9), 152.0 (C4),
150.4 (C2), 147.9 (C3), 135.0 (Co), 132.2 (C7), 131.1 (Cm), 128.9 (C2), 127.0 (Cp), 124.1 (C5), 124.0 (C6), 122.1 (C10), 120.2
(C1), 117.9 (C8), 111.6 (C3), 39.6 (-CH3); FTIR: ṽ = 2802 (w), 2112 (w), 1605 (m), 1591 (m), 1489 (s), 1426 (m), 1406 (s),
1353 (s), 1297 (s), 1253 (m), 1191 (s), 1147 (s), 1112 (s), 1059 (m), 944 (m), 906 (w), 813 (s), 745 (s), 671 (s). HRMS
(ESI) ([BiPh(L)2+Na]+) calcd. for C40H33BiO6N2Na m/z: 869.2040, found: 869.2032; Elemental analysis calcd (%) for
BiC40H33O6N2: C 56.74, H 3.93, N 3.31; found: C 56.62, H 3.84, 3.39.
[BiPh(4-DEAF)2] (3c): The title compound was prepared according to GP2 using 3 (1.0 mmol, 309 mg). Yield: 352 mg
(0.39 mmol, 78 %), orange powder; m.p. 179-180 C; 1H NMR (600 MHz, DMSO-d6): δ 8.67 (d, J = 9.2 Hz, 4H; H2), 8.43
(dd, J = 7.9, 1.1 Hz, 2H; Ho), 8.04 (dd, J = 7.9, 1.3 Hz, 2H; H5), 7.74 (d, J = 8.4 Hz, 2H; H8), 7.69 (ddd, J = 8.4, 6.9, 1.6
Hz, 2H; H7), 7.50 (t, J = 7.6 Hz, 2H; Hm), 7.41 (ddd, J = 7.9, 6.9, 1.3 Hz, 2H; H6), 7.04 (tt, J = 7.3, 1.3 Hz, 1H; Hp), 6.69 (d,
J = 9.2 Hz, 4H; H3), 3.37 (q, J = 7.0 Hz, 6H; -CH2), 1.07 (t, J = 7.0 Hz, 12H; -CH3); 13C NMR (151 MHz, DMSO-d6): δ
176.8 (C4), 153.3 (C9), 152.2 (C4), 147.9 (C2), 147.4 (C3), 135.1(Co), 132.1 (C7), 131.1 (Cm), 129.2 (C2), 127.0 (Cp), 124.1
(C5), 124.0 (C6), 122.2 (C10), 119.2 (C1), 117.9 (C8) 111.0 (C3), 46.6 (-CH2), 12.5 (-CH3); FTIR: ṽ = 2970 (w), 1605 (m),
1590 (m), 1542 (m), 1494 (s), 1479 (m), 1398 (s), 1356 (m), 1303 (m), 1269 (m), 1198 (s), 1155 (m), 1076 (w), 994 (w),
909 (w), 813 (m), 745 (s), 696 (s), 671 (m), HRMS (ESI) ([BiPh(L)2+Na]+) calcd. for C44H41BiO6N2Na m/z: 925.2666, found:
925.2660; Elemental analysis calcd (%) for BiC44H41O6N2: C 58.54, H 4.58, N 3.10; found: C 58.39, H 4.63, N 3.17.
[BiPh(4-MF)2] (4c): The title compound was prepared according to GP2 using 4 (1.0 mmol, 268 mg). Yield: 295 mg (0.36
mmol, 72 %), bright yellow powder; m.p. 225 C (decomp); 1H NMR (600 MHz, DMSO-d6): δ 8.77 (d, J = 9.0 Hz, 4H; H2),
8.43 (d, J = 7.2 Hz, 2H; Ho), 8.07 (dd, J = 8.0, 1.4 Hz, 2H, H5), 7.81 (d, J = 8.4 Hz, 2H, H8), 7.75 (ddd, J = 8.4, 6.9, 1.4 Hz,
2H; H7), 7.52 (t, J = 7.2 Hz, 2H; Hm), 7.45 (ddd, J = 8.0, 6.9, 0.9 Hz, 2H; H6), 7.06 (d, J = 9.0 Hz, 4H; H3), 7.04 (m, 1H;
Hp), 3.86 (s, 3H; O-CH3); 13C NMR (151 MHz, DMSO-d6): δ 178.4 (C4), 153.6 (C9), 151.4 (C4), 150.3 (C2), 148.63 (C3),
135.0 (Co), 132.9 (C7), 131.3 (Cm), 129.0 (C2), 127.1 (Cp), 125.4 (C1), 125.4 (C5), 124.3 (C6), 121.9 (C10), 118.2 (C8),
114.0 (C3), 55.1 (O-CH3); FTIR: ṽ = 3062 (w), 2289 (w), 1607 (m), 1590 (m), 1551 (s), 1518 (m), 1500 (s), 1480 (m), 1463
(m), 1403 (m), 1351 (m), 1300 (s), 1250 (m), 1205 (s), 1176 (m), 1148 (s), 1110 (s), 1025 (m), 995 (s), 907 (s), 830 (s),
814 (m), 752 (s), 726 (m), 705 (m), 671 (m); HRMS (ESI) ([BiPh(L)2+Na]+) calcd. for C38H27BiO8 m/z: 843.1407, found:
843.1390; Elemental analysis calcd (%) for BiC38H27O8: C 55.62, H 3.32; found: C 55.62, H 3.24.
[BiPh(3,4-DMF)2] (5c): The title compound was prepared according to GP2 using 5 (1.0 mmol, 298 mg). Yield: 392 mg
(0.44 mmol, 88 %), bright yellow powder; m.p. 152 C; 1H NMR (600 MHz, DMSO-d6): δ 8.49 (dd, J = 8.58, 1.6 Hz, 2H;
H5), 8.45 (d, J = 7.4 Hz, 2H; Ho), 8.16 (s, 2H; H2), 8.06 (dd, J = 8.1, 1.4 Hz, 2H; H6), 7.38 (d, J = 8.3 Hz, 2H; H8), 7.75
(ddd, J = 8.3, 7.0, 1.6 Hz, 2H; H7), 7.51 (t, J = 7.4 Hz, 2H; Hm), 7.44 (m, 2H; H6), 7.03 (t, J = 7.4 Hz, 1H; Hp), 6.92 (d, J =
8.7 Hz, 2H; H5), 3.81 (s, 6H; 3 O-CH3), 3.72 (s, 6H; 4 O-CH3); 13C NMR (151 MHz, DMSO-d6): δ 178.4 (C4), 153.6 (C9),
150.5 (C4), 149.7 (C3), 148.7 (C2), 148.3 (C3), 134.9 (Co), 132.8 (C7), 131.2 (Cm), 127.1(Cp), 125.4 (C1), 124.2 (C5, C6),
121.9 (C6), 121.6 (C10), 118.3 (C8), 111.3 (C5), 110.4 (C2), 55.4 (3-OCH3), 55.3 (4-OCH3); FTIR: ṽ = 2997 (w), 2832 (w),
1596 (w), 1581 (w), 1557 (m), 1542 (m), 1494 (s), 1457 (m), 1401 (s), 1358 (m), 1311 (m), 1266 (s), 1200 (s), 1178 (s),
1135 (s), 1115 (m), 1029 (m), 1009 (m), 941 (w), 884 (w), 853 (m), 811 (m), 754 (s), 735 (m), 714 (m), 696 (m); HRMS
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(ESI) ([BiPh(L)2+H]+) calcd. for C40H32BiO10 m/z: 881.1799, found: 881.1782; Elemental analysis calcd (%) for BiC40H31O10:
C 54.55, H 3.55; found: C 54.28, H 3.56.
[BiPh(3,4,5-TMF)2] (6c): The title compound was prepared according to GP2 using 6 (1.0 mmol, 328 mg). Yield: 385 mg
(0.41 mmol, 82 %), bright yellow powder; m.p. 184 C; 1H NMR (600 MHz, DMSO-d6): δ 8.47 (dd, J Hz, 2H; H5), 8.07
(dd, J = 8.2, 1.7 Hz, 2H; Ho) 7.95 (s, 4H; H2, H6), 7.83 (dd, J = 8.4, 1.0 Hz, 2H; H8), 7.76 (ddd, J = 8.6, 6.9, 1.6 Hz, 2H;
H7), 7.55 (t, J = 7.6 Hz, 2H; Hm), 7.45 (ddd, J = 8.0, 6.8, 1.1 Hz, 2H; H6), 7.06 (t, J = 7.3 Hz, 1H; Hp), 3.73 (s, 6H; 4 O-
CH3), 3.62 (s, 12H; 3, 5 O-CH3). 13C NMR (151 MHz, DMSO-d6) δ 178.8 (C4), 153.7 (C9), 152.6 (C3, C5), 150.1 (C2),
149.2 (C3), 138.6 (C4), 134.8 (Co), 133.0 (C7), 131.4 (Cm), 127.8 (C1), 127.1 (Cp), 124.3 (C5), 124.2 (C6), 121.8 (C10),
118.4 (C8), 105.1 (C2, C6), 60.1 (4-OCH3), 55.5 (3-OCH3, 5-OCH3); FTIR: ṽ = 2936 (w), 2829 (w), 1735(w), 1578 (m),
1553 (m), 1495 (m), 1401 (s), 1337 (m), 1290 (s), 1240 (m), 1198 (m), 1120 (s), 1056 (m), 1000 (m), 946 (w), 887 (s),
753 (s), 731 (m), 684 (m); HRMS (ESI) ([BiPh(L)2+H]+) calcd. for C42H36BiO12 m/z: 941.2011, found: 941.20049; Elemental
analysis calcd (%) for BiC42H35O12: C 53.63, H 3.75; found: C 53.56, H 3.81.
X-ray crystallography
Crystallographic data of compounds 1b, 2b, and 5b were collected on a Bruker APEX-II CCD diffractometer using
graphite-monochromated MoKα radiation (λ = 0.71073 Å) at 123(1) K. Data was collected and processed with the Bruker
Apex2 v2014.7–1 software, polarisation, Lorentz and absorption corrections (multi-scan-SADBABS)[55] were applied.
Crystallographic data of compounds 2c.(THF), 5c.(DMSO), and 6c.(DMSO) were collected at the MX1 beamline at the
Australian Synchrotron, Melbourne, Victoria, Australia, with synchrotron radiation (17.4 keV, λ = 0.71073 Å). All data was
collected at 100(1) K, maintained using an open flow of nitrogen. XDS was used for data collection and reduction of the
data.[56] Multi‐scan absorption corrections (SADABS) were applied.[57] Crystallographic data of compounds 6b and
1c.(DMSO) were collected on a XtaLAB Synergy, Dualflex, HyPix diffractometer using MoKα (λ = 0.71073 Å) at 123.00(10)
K. Data was processed with the CrysAlisPro version 1.171.40.53 software suite. Empirical absorption corrections were
applied using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm.
All suitable crystals were mounted on a glass fibre loop under oil. All data was solved using Olex2[58] graphical interface
with the ShelXT[59] structure solution program using Intrinsic Phasing. The models were refined on F2 data with the
SHELX[60] refinement package using Least Squares minimisation. All non-hydrogen atoms including disorder fragments
were refined anisotropically. Hydrogen atoms on C atoms were placed at calculated positions (C–H = 0.95–0.98 Å) and
refined using the riding model, with Uiso(H) = 1.2 Ueq(C) for CH and CH2 groups and 1.5 Ueq(C) for CH3 groups. Molecular
graphics were generated using Olex2. A summary of the crystallographic data can be found in Table S2 and S3 of the
supporting information. CCDC 1970402, 1971006, 1975036, 1977219, 1977244, 1971258, 1971300 and 1970685 contain
the supplementary crystallographic data for this paper. These data are provided free of charge by The Cambridge
Crystallographic Data Centre.
DMEM cell culture medium stability studies
The stability of complexes 6b and 6c was assessed in DMEM containing 10% (v/v) FBS and 1% (v/v)
penicillin/streptomycin antibiotics and DMEM containing 1% antibiotics only. Stock solutions of 6b and 6c were prepared
in sterile DMSO and were diluted in the cell culture media (final DMSO concentration: 0.8 %). UV-Vis spectra were
obtained on an Agilent Cary 60 UV-Vis spectrophotometer scanning in the range 280-750 nm.
Bacterial cultures
Methicillin resistant S. aureus (M118797), vancomycin resistant E. faecalis (M846910), S. aureus (A134/ATCC6538), E.
coli (G102) and P. aeruginosa (ATCC27853) were recovered from frozen (-80 oC) glycerol stocks (15% v/v) on either
nutrient agar (MRSA, VRE, S. aureus) or Luria-Bertani (LB) agar (E. coli, P. aeruginosa) at 37 oC for 24 hours. Single
colonies were then selected and incubated (37 oC) for a further 18 hours whilst shaking at 180 rpm, in either 3 mL of Brain
Heart Infusion (BHI) media (Gram-positive bacteria) or LB media (E. coli, P. aeruginosa).
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10.1002/chem.202000562
Chemistry - A European Journal
Bacterial MIC determination
The Minimum Inhibition Concentration (MIC) of flavonols 1–6 and Bi-compounds 2b–6b and 1c–6c was determined using
a broth micro-dilution method according to Clinical Standard Laboratory Institute (CSLI) guidelines. Briefly, 2.5 L of a
one in ten dilution of overnight cultures of bacteria (approximately 5 x 105 CFU/mL) were added to falcon tubes containing
1 mL of serially diluted bismuth compounds in the concentration range 50-0.78 g/mL in either BHI or LB media. Cultures
containing no added compounds served as a control. The MIC was determined as the lowest concentration of compound
that inhibited the visible growth of the bacteria after incubating (37 oC, 180 rpm) for 20 hours.
COS-7 Cell culture
COS-7 (monkey kidney fibroblast) cells were maintained as cell monolayers at 37 C in 5% CO2 in Dulbecco’s modified
Eagle’s medium (DMEM) with the addition of 10% heat inactivated fetal bovine serum (FBS), 1% Pen Strep, and 1%
GlutaMAX. The cells were passaged every 3 or 4 days. Cells in the exponential growth phase were used for subsequent
assay and uptake experiments.
Cell viability assay
The effect of the prepared Bi-compounds and free flavonols on COS-7 cell viability was assessed using the CellTitre-Blue
assay (Promega) according to the manufacturer’s instructions. Stock solutions of the bismuth complexes, parent flavonols,
and BiPh3 were prepared in sterile DMSO to give working solutions of 5-10 mM, and were diluted in fresh cell culture
medium to give a concentration range of 50.0-2.1 M (final DMSO concentration in all cases: < 0.1 % v/v). COS-7 cells
were seeded into 96-well plates (10,000 cells/well in 100 L complete cell media) and incubated for 24 hours at 37 C/5%
CO2. The cell media was then removed the and replaced with 100 L of drug/media solution in the concentration range
of 50.0-2.1 M, in duplicate rows for each compound. COS-7 cells treated with fresh cell media served as an untreated
control. The cells were incubated for 24 h, followed by the addition of CellTitre-Blue (20 L). Wells containing only 100 L
of cell culture media and 20 L of CellTitre-Blue served as a negative (no cells) control. The cells were further incubated
for 4 h. The fluorescence intensity of the wells was then measured on a BMG LABTECH CLARIOstar microplate reader
using excitation of 544 nm and emission of 590 nm. The fluorescence intensity of the negative control was subtracted
from all readings as a background fluorescence value. The IC50 values were calculated in Graphpad Prism 7 by non-linear
regression (curve-fit), log(inhibitor) vs normalised response. The IC50 values are presented as the mean result from at
least three independent experiments.
Cellular uptake in COS-7 cells: COS-7 cells were seeded (5  105 cells/well in 2.5 mL cell culture medium) into 6-well
plates and incubated for 24 h at 37 C/5% CO2 prior to compound exposure. Stock solutions of the Bi-compounds were
prepared in sterile DMSO and diluted in cell culture medium to 5 M immediately before use (final DMSO concentration:
0.1 % v/v). The old culture medium was removed from the wells and replaced with 2.5 mL of medium containing the
respective Bi-compound. Triplicate wells for each Bi-compound were prepared (one row per plate). An untreated cells
control containing fresh cell medium and vehicle (DMSO) control containing 5 M DMSO in cell culture media were
processed on the same plate in triplicate. The plates were then incubated for 24 h at 37 C/5% CO2. The cell culture
solutions were then aspirated from the wells and the cells were washed with PBS solution (2 mL  3). Trypsin (400 L)
was added to each well and the plates were incubated for 5 minutes to detach the cells. The trypsin was neutralized by
the addition of 4 mL of fresh cell culture medium, and the contents of each well were transferred to 15 mL centrifuge
tubes. The tubes were centrifuged for 5 minutes at 1600 rpm and the culture medium supernatant was discarded. PBS
(1.5 mL) was added to the tubes and were subsequently vortexed to wash the cells and then centrifuged at 5 minutes at
1600 rpm. This washing procedure was repeated three times. The number of cells in each sample was then counted with
a hemocytometer. The cell pellets were stored in a -80 C freezer prior to HNO3 digestion and ICP-MS analysis. Three
independent experiments were performed.
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10.1002/chem.202000562
Chemistry - A European Journal
Cellular uptake in VRE: Overnight cultured VRE (diluted to 1 x 106 CFU/mL) was added to centrifuge tubes containing
the Bi-compounds diluted in BHI so that the overall concentration of the complexes was 5 g/mL. After incubating for 20
hours (37 oC, 180 rpm), the falcon tubes were centrifuged for 15 minutes at 4000 rpm, with the resulting supernatant
discarded. The pelleted cells were then washed 6x with 1 mL PBS before being re-suspended in 3 mL BHI in order to
determine the CFU/mL of bacteria present (serially diluted and overnight incubation of nutrient agar plates). The centrifuge
tubes were then re-centrifuged (15 minutes, 4000 rpm) and washed a further 6x with 1 mL PBS. The cell pellets were
then stored in a -80 C freezer prior to HNO3 digestion and ICP-MS analysis.
Cell pellet preparation for ICP-MS: Concentrated (70%) HNO3 (200 L) were added to the COS-7 or VRE cell pellets
and were then incubated in a 60 C water bath for 24 h. The digested samples were then diluted with 4.8 mL HNO3 (3%)
to obtain an approximately 3% HNO3 sample solution. The Bi content in each sample was determined by ICP-MS on a
PerkinElmer NexION 350 ICP-MS measuring Bi at 208.98 amu. Raw count rates from the analyses were externally
standardised by means of a calibration curve constructed using a commercially available Bi stock solution and an internal
standard of 80 ppb Pb was used. Precision is of the order of 3.2 % RSD from the single analyses. The final %Bi calculation
is presented as the mean result from at least three independent experiments with standard error calculated.
Acknowledgments
We thank the Australian Research Council (DP170103624) and Monash University for financial support for this research.
The authors acknowledge that part of this research was undertaken on the MX1 beamline at the Australian Synchrotron
(Melbourne, Australia), part of ANSTO. We would like to thank Rajini Brammananth (Department of Microbiology, Monash)
for performing initial bacterial measurements, and Dr Craig Forsyth and Michael Stevens for their assistance with the
crystallography.
Conflicts of interest
The authors declare no conflicts of interest.
Keywords: bismuth • flavonols • structure • antibacterial activity • cellular uptake
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